High-cell density shake-flask expression and rapid purification of the large fragment of Thermus aquaticus DNA polymerase I using a new chemically and temperature inducible expression plasmid in Escherichia coli.
We have developed a new expression vector, pcI(ts) ind(+), based upon the powerful rightward promoter of bacteriophage lambda, which is controlled by a temperature-sensitive and chemically-inducible version of the lambda repressor on the same plasmid. Locating the repressor gene on the plasmid makes this vector "portable" in that it can be used to transform any strain of Escherichia coli. Hence, control over strains, induction conditions, and harvest times can be used to optimize yields of heterologous proteins. To provide a proof of concept, we show that E. coli recA(+) and recA(-) host cells transformed with pcI(ts) ind(+) modKlenTaq1 (a modified version of the large fragment of Thermus aquaticus DNA polymerase I) could be grown to high cell densities in multiple shake-flasks. A mutant version of modKlenTaq1 (V649C) could be induced by simply raising the thermostat setting from 30 to 37 degrees C and (in the case of recA(+) cells) adding nalidixic acid to achieve full induction (12-13% of the total cellular protein). Using a rapid, two-step purification process, it was possible to purify nearly 300 mg of modKlenTaq1 V649C from six 2.8-L baffle-bottomed shake-flasks each holding 1.5L of culture for a final yield of approximately 33 mg per liter or 3mg of purified enzyme per gram of cells wet weight.
Brandis
John W
JW
Institute for Cell and Molecular Biology, 2500 Speedway, University of Texas at Austin, Austin, TX 78712, USA. jwbrandis@mail.utexas.edu
Johnson
Kenneth A
KA
D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D004256:DNA Polymerase I / Q000096:biosynthesis* / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D010957:Plasmids / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019914:Taq Polymerase / Q000096:biosynthesis* / Q000302:isolation & purification*; D013824:Thermus / Q000201:enzymology
D019914:Taq Polymerase; D004256:DNA Polymerase I